Background: Immune checkpoint inhibitors have been widely used in the treatment of advanced lung cancer, but immunotherapy still shows poor efficacy or leads to hyperprogression in some patients. Some studies demonstrated that EGFR mutations, ALK fusion, interferon pathway gene mutation, and Wnt signal pathway mutation might reduce the efficacy of immunotherapy and lead to tumor hyperprogression. The aim of this study was to determine the frequency of gene variations associated with immunotherapy resistance or hyperprogression in patients with advanced lung cancer. Methods: The tumor tissues or blood samples of 129 patients with advanced lung cancer were collected. The tumor tissues were sequenced by targated next-generation sequencing (55 genes, Illumina novaseq 6000), MSI (PCR), and PD-L1 detection (IHC). Blood cfdna was extracted and sequenced by targeted next-generation sequencing (55 genes, Illumina novaseq 6000). Results: A total of 129 patients with advanced lung cancer were included in the study, including 126 patients with non-small cell lung cancer and 3 patients with small cell lung cancer. Tissue was available for testing in 113 patients, blood samples were collected from 106 patients, and cerebrospinal fluid samples were collected from 1 patient. Among 85 samples tested for MSI, there were 81 cases of MSS (95.3%), 3 cases of MSI-L(3.5%), and 1 case of MSI-H (1.2%). PD-L1 expression was detected in 60 samples. 23 samples (38.3%) had PD-L1 expression < 1%, 31 samples (51.7%) had PD-L1 expression greater than 1% but less than 49%, and 6 samples (10%) had PD-L1 expression > 50%. Among the genes related to immunotherapy, the mutations of genes related to cell growth or cell cycle regulation (EGFR, ALK, CDKN2A, CCND1) accounted for 86 (66.7%), 12 (9.3%), 15 (11.6%), 8 (6.2%), respectively. The mutation of antigen presentation related gene (B2M) accounted for 2 (1.6%). The mutations of interferon signaling pathway related genes (JAK1, JAK2, JAK3, IFNGR1, PIAS4 and SOCS1) accounted for 6 (4.7%), 7 (5.4%), 3 (2.3%), 3 (2.3%), 1 (0.8%) and 1 (0.8%), respectively. Wnt signaling pathway related genes (APC, AXIN1, CTNNB1) accounted for 8 (6.2%), 2 (1.6%) and 4 (3.1%). Mutations in tumor suppressor genes (PTEN, STK11, KEAP1, PTPN2) accounted for 4 (3.1%), 8 (6.2%), 6 (4.7%) and 3 (2.3%). Mutations in other genes (APLNR, DNMT3A, MDM2, MDM4) accounted for 3 (2.3%), 1 (0.8%), 7 (5.4%) and 1 (0.8%). Conclusions: Our study shows that in patients with advanced lung cancer, there are multiple gene variants related to drug resistance or hyperprogression of immune checkpoint inhibitors, suggesting that this may be the reason for the difference in immunotherapy efficacy in patients. It is necessary to further study the efficacy of immunotherapy in patients with different gene mutations and the combination of immune checkpoint inhibitors for patients with different pathway mutations.